Treatment of pigmented tissues using optical energy

ABSTRACT

A method and apparatus for selectively photobleaching or killing pigmented tissues by photochemically converting pigments in the tissues using light and specifically two-photon excitation. Phototoxic products thereby produced then kill pigmented cells. Hyperthermia or an exogenous agent can also be added to augment efficacy. The present invention is also directed to selective thermal destruction of pigmented tissues using related optical means.

This application is a continuation-in-part of U.S. patent applicationSer. No. 09/130,213 filed Aug. 6, 1998 which is a continuation-in-partof U.S. patent application Ser. No. 08/739,801, filed on Oct. 30, 1996.

BACKGROUND OF THE INVENTION

The present invention is directed to a method and apparatus for treatingpigmented tissues by selective photoactivation of pigments in suchtissues using optical energy and more specifically two-photonexcitation. This selective photoactivation may be used to effectphotobleaching of such pigments or to effect photochemical conversion ofsuch pigments into phototoxic products. Photobleaching reduces oreliminates undesirable pigmentation, for example that caused by pigmentspresent in moles, freckles, hair follicles and tattoos. Photochemicalconversion produces phototoxic products that destroy pigmented tissues,such as those pigmented tissues in pigmented tumors. The presentinvention is also directed to selective thermal destruction of pigmentedtissues using related optical means.

Photobleaching is the transient or permanent reduction of pigmentationin pigmented tissues upon optical illumination, typically occurringduring intense illumination with visible or ultraviolet light.Photobleaching occurs when photoactive pigments are photochemicallytransformed from a highly colored state to a less highly colored state(de-pigmentation). For example, photobleaching may be used to reduce oreliminate undesirable pigmentation present in moles and hair folliclesor to destroy dyes present in tattoos. It is desired that treatedtissues will exhibit localized de-pigmentation without side effects,such as irritation or cell necrosis. However, previous methods forphotobleaching tissues using visible or ultraviolet light have producedundesirable collateral effects, including irritation of surroundingtissues and possible scarring at the treatment site.

In contrast to photobleaching, photochemical conversion of pigments intophototoxic products involves stimulation of localized cell necrosis intreated tissues. This is also effected by optical illumination,typically occurring when intense visible or ultraviolet light is used toilluminated susceptible pigmented tissues. Such localized necrosis maybe useful for selective destruction of diseased tissues, such as thosepresent in tumors or benign skin lesions.

More specifically, an important subset of pigmented tissues arepigmented tumors, such as melanomas, which are life threatening andhighly difficult to treat. While melanomas can be treated if detectedearly using standard surgical, radiation or chemotherapeutic methods,these methods still do not have acceptable levels of effectiveness andproduce high levels of collateral damage to normal tissue. Hence, evenif detected relatively early, the prognosis is usually poor.

Further, if a melanoma has metastasized beyond the primary tumor site,less than 20% of patients will survive beyond five years. For suchmelanomas, there are no effective therapies. Patients diagnosed withsuch a metastatic melanoma will survive on average only 3-6 months afterthe diagnosis even with therapeutic intervention.

Further exacerbating the difficulties in treating melanomas is the factthat the incidence of melanoma in Caucasians is increasing at a rate of6% per year. This is currently the second fastest rate of increase incancer occurrences—second only to tobacco related cancers of the lung inwomen. Currently, the lifetime risk of melanoma in the U.S. is 1 in 75.Accordingly, new effective therapeutic modalities are required to treatboth primary and metastatic pigmented tumors such as melanomas.

One possible approach for treating pigmented tissues involves the use ofmelanins, their precursors, and other endogenous or exogenous pigments.

More specifically, there are several pigments in humans that arecollectively known as melanins. The function of melanins are to protecttissues from the deleterious effects of electromagnetic radiation (e.g.light). However, melanins and their precursors can also be converted tophototoxic products. For example, a melanin precursor (5-SCD) has beenshown to photobind to DNA after exposure to 300 mn (ultraviolet light)illumination. Further, 5-SCD has been shown to be chemically unstable inthe presence of ultraviolet (UV) illumination and oxygen, therebysuggesting that phototoxic products of the (1) Type I variety(phototoxic) or the (2) Type II variety (photocatalytic) may beproduced.

Additionally, many melanoma cells are amelanotic. These cells producemelanin precursors but only small quantities of melanin. Phototoxicdamage (induction of single strand breaks) to DNA by at least twoprecursors to melanin (5-SCD and DIHCA) has been demonstrated uponexposure to UV light. Amelanotic cells will be killed by photodynamictherapy (PDT) performed on such precursors to melanin (e.g., 5-SCD,DIHEA). Thus, melanomas can be killed by delivering energy via light.

However, utilization of such phototoxic reactions by illumination ofmelanin, melanin precursors, or other endogenous pigments has notpreviously been possible. The UV/Near UV light required forphotoactivation is unable to penetrate into normal or cancerous skin(i.e. beyond 2-3 mm.) More specifically, the poor penetration of suchlight has produced little effect on patients whose skin tumors arelarger than or at a depth greater than 3 mm. As a result, only 40-50% ofpatients whose tumors exceed 3 mm will survive. Accordingly, thesurvival rate of melanoma patients with tumors whose depth is less than1 mm is drastically better than those who have tumors which are eitherlocated at a depth of greater than 3 mm or extend to a depth greaterthan 3 mm.

Previous photodynamic methods using UV/Near UV light also producedundesirable collateral effects that not only prohibited thephotoconversion of melanin and prevented it from killing pigmentedtissues but also was potentially dangerous to the patient. For example,UV light can create thymidine dimers which damage genetic material. DNAdamage is a major and possibly the sole cause of skin cancers likemelanomas. Melanin's absorbance of UV light is designed to prevent thisfrom happening. However, UV light, chemotherapy, and ionizing radiationhave recently been shown to increase the virulence of tumor cells. As aresult, tumor cells when treated with UV light will have a greatermutation and error rate because the UV light can inactivate mechanismsdesigned to identify and correct genetic errors (in addition to creatingnew errors). Therefore, prior techniques were not only unable toeffectively kill pigmented tissues by accessing endogenous pigments butalso created side effects that could be lethal.

In many instances, the effectiveness of various photodynamic processeshave been found to be markedly increased by simultaneous photoactivationand localized heating (hyperthermia). Typically, by heating thetreatment zone 2-10° C. above normal temperatures, the effectiveness ofPDT is increased many fold. Such heating alone, however, has not beenshown to produce a significant therapeutic effect. In contrast, theinventors of the present invention have conceived that more acutelocalized heating (i.e., >2-10° C. temperature rise) of tissues andtissue components within the treatment zone may produce a therapeuticeffect by causing thermal overload in the treated tissues.

Therefore, it is an object of the present invention to provide a methodfor accessing endogenous pigments in pigmented tissues so as to be ableto selectively photobleach said pigments.

It is another object of the present invention to provide a method foraccessing endogenous pigments in pigmented tissues so as to be able tophotochemically convert said pigments into phototoxic products.

It is another object of the present invention to provide a method thatwill access said endogenous pigments in pigmented tissues withoutaccessing endogenous pigments in healthy tissues surrounding saidpigmented tissues.

It is another object of the present invention to provide a method thatwill augment the effectiveness of said photochemical conversion of saidendogenous pigments in said pigmented tissues through the localizedapplication of hyperthermia in said pigmented tissues.

It is another object of the present invention to provide a method thatwill photothermally destroy pigmented tissues without harming healthytissues surrounding said pigmented tissues.

SUMMARY OF THE INVENTION

The present invention is directed to a method and apparatus fortreatment of a particular volume of tissue or material containing anendogenous pigment. In general, typically, the present invention usesthe unique properties of simultaneous two-photon excitation withendogenous pigment in a particular volume of tissue, such as a tumor, toselectively photoactivate the pigment.

This photoactivated pigment may thereby be photobleached orphotochemically converted into a phototoxic product. Suchphotoactivation results from the simultaneous two-photon excitation ofthe pigment. Preferably, the photons responsible for photoactivation areprovided by a laser which produces a beam of light comprising a train ofone or more ultrashort pulses. This beam of light can be a focused beamof light if the location and extent of the particular volume of tissueto be treated is precisely known. The focused beam of light can then bescanned throughout the volume of the tissue to treat the entirety of thepigmented tissue. Alternatively, where the location and extent of thepigmented tissue in a volume of tissue is not precisely known, anon-focused light beam can be used.

In an alternative embodiment, an exogenous photodynamic agent can beadded to the particular volume of tissue. The exogenous agent can bephotoactivated by the simultaneous two-photon excitation. Activation ofthe exogenous photodynamic agent augments the effectiveness of theendogenous pigment.

In a further alternate embodiment of the invention, the effectiveness ofsuch photoactivation is augmented through the localized application ofhyperthermia in the pigmented tissues.

In an additional further alternative embodiment of the invention, theparticular volume of tissue is treated with light to promote thermaloverload of the pigmented tissues. Thermal overload heats and kills thepigmented tissues.

BRIEF DESCRIPTION OF THE DRAWINGS

In describing the preferred embodiments, reference is made to theaccompanying drawings:

FIG. 1 illustrates an example energy level diagram for simultaneoustwo-photon excitation;

FIG. 2 illustrates an example of absorption and scattering propertiesfor animal tissue covering the ultraviolet to infrared spectral region;

FIG. 3 shows the general trends in optical absorption properties ofanimal tissue for short wavelength and long wavelength light;

FIG. 4 illustrates a comparison of optical activation in tissue whensingle-photon and two-photon excitation methods are used;

FIG. 5 illustrates an embodiment of the present invention for selectivetwo-photon photoactivation of melanin, melanin-precursors or endogenouspigments using focused light;

FIG. 6 illustrates an another embodiment for selective two-photonphotoactivation of melanin, melanin-precursors, or endogenous pigmentsusing focused light;

FIG. 7 illustrates a further embodiment for selective two-photonphotoactivation of melanin, melanin-precursors, or endogenous pigmentsusing non-focused light;

FIG. 8 illustrate still another embodiment for selective two-photonphotoactivation of melanin, melanin-precursors, or endogenous pigmentsin a subsurface tissue using non-focused light;

FIG. 9 illustrates an alternate embodiment for the present inventionwherein a focused light beam is used to thermally overload and killpigmented tumor cells; and

FIG. 10 illustrates another alternate embodiment for the presentinvention wherein a non-focused light beam is used to thermally overloadand kill pigmented tumor cells.

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENT

The present invention is directed to a method and apparatus for treatingpigmented tissues using light. Such treatment includes the followingphotochemical outcomes of therapeutic value: (1) the elimination ofundesirable pigmentation in pigmented tissues through photobleaching;and (2) the permanent destruction of pigmented tissues throughphotochemical conversion of pigments into phototoxic products. Morespecifically, simultaneous two-photon excitation is used tophotochemically convert endogenous or exogenous pigments into desiredphotoactive products, resulting in the desired photobleaching or tissuedestruction. Photobleaching is used to reduce or eliminate undesirablecoloration of tissue, such as that in moles, freckles, hair folliclesand tattoos. The production of phototoxic products may be used topreferentially kill pigmented tumor cells or other undesirable tissueswhile sparing normal cells. Significantly, the methods and apparatus inthe present invention used for photobleaching and production ofphototoxic products utilize equivalent photoactivation mechanisms,differing substantially only in the intended treatment target.

In the preferred embodiment, the present invention uses simultaneoustwo-photon excitation to photoactivate pigments in the pigmentedtissues, yielding photobleached or phototoxic products.

In an alternate preferred embodiment, the present invention uses relatedoptical means to selectively destroy pigmented tissues via photothermalmeans.

Simultaneous Two Photon Excitation

“Simultaneous two-photon excitation” is the non-linear opticalexcitation occurring as a result of the essentially simultaneousinteraction of two photons originating from a single ultrashort laserpulse with one or more agents or pigments to produce one or morephotoactivated agents or pigments. “Non-linear optical excitation” meansthose excitation processes involving the essentially simultaneousinteraction of two photons with one or more agents or pigments.“Essentially simultaneous interaction” means those excitation processesoccurring as a result of the interaction of one or more agents orpigments with photons provided by a single ultrashort laser pulse.Ultrashort means less than approximately 10 ns.

As shown in FIG. 1, simultaneous two-photon excitation to an allowedenergy level 10 occurs when a photoactive agent is excited from a firstallowed electronic energy level 16 upon absorption of a certain energyE₁ that is provided by the simultaneous, combined interaction of twophotons 12 and 14 with the agent. If the energies of both photons 12 and14 are identical, the excitation process is termed “degenerate”. Thesimultaneous interaction of the two photons is frequently described asbeing mediated by a transient virtual state 20 with a lifetime on theorder of 10 femtoseconds (fs) or less. If both photons do not interactwith the agent during this lifetime, excitation does not occur and theagent fails to reach the excited state S_(n) (18). Typically,intersystem crossing, IX, subsequently occurs to bring the excited agentto a long-lived activated state T_(m) from which a photochemicalreaction R can occur.

Simultaneous two-photon excitation may thereby be used to exciteprocesses that normally occur upon absorption of a single UV or visiblephoton through the simultaneous absorption of two near-infrared photons.

An example of the simultaneous two-photon excitation process is thepromotion of melanin precursors from a ground electronic state to anexcited electronic state through the simultaneous absorption of twophotons at 600 nm, followed by binding of the excited melanin precursorto DNA (this is conventionally excited using a single photon at 300 nm).In this example, the probability of excitation is related to the productof the instantaneous or peak powers of the first of two photons 12 andthe second of two photons 14. This can be conceptualized in the form ofa photochemical reaction,Molecule_(GROUND STATE)+2 hν₆₀₀ →Molecule_(EXCITED STATE)   (1)which shows that a molecule in the ground state is promoted to anexcited state following simultaneous absorption of two photons at 600nm, hν_(600 nm). The reaction rate R, is given by R=k[Molecule_(GROUND STATE)] [hν_(600 nm)]², where k is a rate constant andwhere [Molecule_(GROUND STATE)] and [hν₆₀₀] symbolize concentrations ofground state molecules and excitation photons, respectively. Hence, dueto the well known quadratic dependence on instantaneous photonirradiance, simultaneous two-photon excitation to an allowed energylevel 10 is also referred to as a non-linear excitation process.

A more detailed explanation of simultaneous two-photon excitation andother non-linear and linear processes is described in U.S. patentapplication Ser. No. 08/739,801 filed Oct. 30, 1996 for “Method ForImproved Selectivity In Photoactivation Of Molecular Agents” assigned tothe same assignee of the present application and which is incorporatedherein by reference.

Significance of Absorbance and Scattering Properties in Single-Photonand Simultaneous Two-Photon Processes:

While the cross-section for simultaneous two-photon excitation may beconsiderably lower than that observed with single-photon excitation, useof the simultaneous two-photon excitation in the present invention maybe favorable over single-photon excitation under many conditions becauseof lower matrix absorption and optical scattering of longer wavelengthoptical radiation. For example, FIG. 2 shows the absorption andscattering properties for various components of animal tissue, such ashuman dermis, covering the ultraviolet (UV) to near infrared (NIR)spectral region.

Specifically; FIG. 2 demonstrates how higher-energy photons 32 mayexperience considerably greater tissue absorption than lower-energyphotons 34. For example, human skin strongly absorbs higher-energyphotons 32 at 400 nm, but is relatively transparent to lower-energyphotons 34 at 800 nm. This is a consequence of the natural absorbance ofhigher-energy photons 32 by blood, pigments, proteins, and geneticmaterials, among other natural components, of skin.

FIG. 2 further demonstrates how higher-energy photons 42 may experienceconsiderably greater tissue scatter than lower-energy photons 44. Anyoptically dense medium, such as human skin, will strongly scatterhigher-energy photons 42, for example at 400 nm, but will exhibit muchlower scatter for lower-energy photons 44 at 800 nm.

These differences in optical properties have two important consequences.First, absorption of short-wavelength, higher-energy photons 32 bytissue can result in undesirable tissue damage upon exposure to UV orother high-energy light. In contrast, negligible effects may beexperienced upon illumination with lower-energy photons 34, such as NIRlight, even when the optical power of the NIR light is many-fold higherthan that of the UV light. Secondly, the inherently high absorption andscatter of higher-energy photons 32 by tissue can result in very shallowtissue penetration depths, while lower-energy photons 34 generally havemuch greater penetration depths.

These important differences in absorption and penetration depthproperties for higher-energy and lower-energy light are shownschematically in FIG. 3. When UV light 50, for example light at 400 nm,impinges on human tissue 52, the majority of the optical energy isimmediately absorbed and scattered in the outermost layers 54, such asthe epidermis and dermis. Absorption may occur due to excitation ofcertain molecules in the cells of these outermost layers 54, such asthose composing the genetic material in the cellular nucleus. Thisabsorption of higher-energy light by cellular constituents can therebyinitiate a variety of collateral photochemical changes 56 in thesecells. These collateral photochemical changes 56 resulting fromabsorption of UV light 50 can include irreversible genetic damage andinduction of cancer.

In contrast, NIR light 58, for example at 800 nm, will not beappreciably absorbed or scattered by tissue 52 or its outermost layers54. The overall depth of penetration will be much greater, and theextent of collateral damage to cells will be substantially lower. Hence,if long-wavelength excitation light is used to replace the higher-energylight used for conventional single-photon excitation, it is possible tophotoactivate specific molecules or pigments using relativelynon-damaging, high penetration depth, simultaneous two-photonexcitation.

Furthermore, the properties of simultaneous two-photon excitation haveadditional implications when coupled with the inherent non-damagingnature and low absorption of NIR light. For example, FIG. 4 compares theextent of optically-induced damage in tissue when single-photonexcitation 60 and simultaneous two-photon NIR excitation 62 methods areused to illuminate a subcutaneous tumor 64.

Single-photon excitation 60 produces a photoactivation zone 66 thatextends substantially along the entire optical path and has nosignificant biospecificity. Hence, in addition to induction of thedesired photoactivation in the tumor 64, collateral damage can occurthroughout surrounding tissues, such as the dermis 68 and surroundinghealthy tissue 70. If the single-photon excitation 60 is focussed, thephotoactivation zone 66 will be slightly enhanced at the focus 72. Thisphotoactivation zone 66, however, might not even extend into the tumor64 if the UV or visible light is absorbed by the epidermis, dermis 68 orsurrounding healthy tissue 70 prior to reaching the tumor 64. This canoccur as a consequence of the inherently high absorptivity of tissue atshort wavelengths.

In contrast, use of NIR light for simultaneous two-photon excitation 62produces a sharply defined remote photoactivation zone 74 that isspatially localized at the focus 76 as a consequence of the non-linearproperties of this excitation method. Such localization of activation insuch a focal zone is a unique property of non-linear excitationprocesses, such as two-photon excitation. Furthermore, because tissuedoes not appreciably absorb NIR light, collateral damage to thesurrounding dermis 68 and healthy tissue 70 is minimized.

Therapeutic Applications of Simultaneous Two-Photon Excitation:

The foregoing discussion suggests that the fundamental differences inthe absorption of UV and NIR light by tissue and cellular constituents,coupled with the special non-linear properties of simultaneoustwo-photon excitation, have direct applicability for improvements invarious medical treatments, specifically in the modification orelimination of pigmented tissues.

Such simultaneous two-photon excitation enables improved localization inthe photoactivation of photoactive agents with significantly reducedpotential for collateral tissue damage compared with that possible usingconventional methods.

Where control of penetration is not critical, non-focussed NIR light maybe used to stimulate simultaneous two-photon photoactivation of agentspresent in a relatively large illuminated area. In such a case, theextent of agent photoactivation is controlled by varying the location,intensity and duration of exposure of such agents to the NIR beam.

Where precise control of penetration depth or volume extent oftherapeutic application is more critical, focussed NIR light may be usedto stimulate the simultaneous two-photon photoactivation process. Insuch a case, beam irradiance, exposure duration, and degree of focussingare used to control the extent of agent photoactivation.

In both cases, high-irradiance NIR light may be used to achieve maximumefficacy. Furthermore, the high penetration depths achievable with NIRlight combined with the inherent localization of photoactivation that ispossible with focused simultaneous two-photon excitation provide a meansfor photoactivating agents in subsurface tissues without damagingoverlying or underlying healthy tissues.

Simultaneous Two-Photon Treatment with Endogenous Pigments

The method of the present invention improves on the above-describedadvantages through the use of simultaneous two-photon excitation toproduce a therapeutic outcome based on photoactivation of endogenouspigments in order to treat pigmented tissues. “Endogenous” meanspre-existing in a patient or target. “Pigments” means naturallyoccurring agents that absorb optical energy. Examples of such pigmentsinclude melanin, melanin precursors, carotenes, porphyrins (such ashemoglobin), various tattoo dyes and other optically active species.“Therapeutic outcome” means photobleaching or photodynamic destructionof treated pigmented tissues resulting from the natural biologicalaction of a photoactivated endogenous pigment. “Photobleaching” is thereduction or elimination of undesirable pigmentation, for example thatcaused by endogenous pigments present in moles, freckles, hair folliclesand tattoos. “Photodynamic destruction” is localized tissue necrosisresulting from photochemical production of phototoxic products thatdestroy pigmented tissues, such as those pigmented tissues in pigmentedtumors. Tissues suitable for treatment include pigmented tissues inwhich a specific therapeutic outcome is desired, such as moles,freckles, pigmented tumors, benign lesions, hair follicles and tattoos.

In a further embodiment of the present invention, a precursor to theendogenous pigments may be used. Examples of such precursors to pigmentsinclude 5-S-cysteinyldopa (5-SCD) and 5,6-dihydroxyindole (DHI), dopa,dopa semiquinone, leucodopachrome, dopachrome, eumalanins, pheomelanins,sepia melanins, and 5,6-dihydroxyindole-2-carboxylic acid. Suchprecursors have both photoprotective and phototoxic abilities. Ametabolic precursor to melanin is a biochemical (e.g. 5-SCD, DHI) thatis produced by the cell as part of the synthetic pathway that producesmelanin. Melanin precursors, when activated by light, can generatephotoxic products that damage cellular materials (e.g., DNA) killing thetarget cells. Melanin precursors can be activated by two-photonexcitation, as explained supra.

As also explained supra, melanin, melanin precursors, and otherendogenous pigments are naturally occurring in human tissue, includingin tumors. Such melanins, melanin precursors, or other endogenouspigments can be converted to phototoxic products after exposure tolight.

The present invention uses the above-described simultaneous two-photonexcitation to specifically target melanin, melanin precursors, or otherendogenous pigments in pigmented tissues (such as melanomas and othertumors). The pigment is converted to a phototoxic product by NIR lightupon simultaneous two-photon excitation. The phototoxic product thencauses damage to the pigmented tissues (by for example photobinding tocellular DNA or causing breaks in this DNA). This kills the cells in thepigmented tissues and, therefore, destroys it. Because simultaneoustwo-photon excitation is used to specifically target the melanin,melanin precursors, or other endogenous pigments only in the targetedtissue, any melanin, melanin precursors, or other endogenous pigments inthe tissue surrounding the targeted tissue are not converted tophototoxic products.

More specifically, use of simultaneous two-photon excitation produces asharply defined focal zone that is substantially localized in depth andcross-section. This focal zone can be localized to the targeted tissue(such as a tumor) to be killed or a small zone within or surroundingthis tissue. As a result, photoactivation will only occur in the focalzone (i.e. in the tumor). Hence, any melanin, melanin precursors, orother endogenous pigment not in the targeted tissue, such as forexample, in tissue surrounding a tumor, will not be photoactivatedbecause it is outside the focal zone.

Additionally, as explained supra, the simultaneous two-photon excitationis able to penetrate deep into normal or cancerous tissue andphotoactivate melanin or other endogenous pigments located deep withinthe tissue. As a result, tumors located deep within the body or large,deep tumors can be reached and destroyed. Destruction of these tumorscan be done without activating melanin or other endogenous pigmentsalong the path of the light or surrounding the tumor.

In addition to photodynamic destruction of pigmented tissues, such asthose in pigmented tumors, the above-described unique features ofsimultaneous two-photon excitation may be used to achieve improvedsafety and specificity in the photobleaching of pigmented tissues, suchas in moles, freckles, hair follicles and tattoos. The pigments presentin such tissues can be activated by simultaneous two-photon activation,as explained supra, and upon activation may become photobleached. Thus,the present invention also uses simultaneous two-photon excitation tospecifically target endogenous pigments in such pigmented tissues,thereby causing photobleaching and a desired reduction or elimination ofapparent pigmentation.

It is a specific preferred embodiment of the present invention to employthe output of a NIR source, such as the mode-locked titanium:sapphirelaser, to induce simultaneous two-photon photoactivation so as tophotoactivate melanin, melanin precursors, or other endogenous pigmentsusing light at a wavelength approximately twice that necessary for suchconversion using conventional single-photon photoactivation. Asexplained supra, such NIR light exhibits improved penetration intotissue relative to that used for conventional single-photonphotoactivation, and is less likely to produce collateral damage intissues adjacent to the desired treatment target.

For the sake of simplicity and clarity, the following descriptions ofpreferred embodiments will focus on photodynamic destruction ofpigmented tumor tissues, such as those in melanomas. However, it isimportant to note that the methods and apparatus described are equallyapplicable to the photobleaching of pigmented tissues, such as moles ortattoos, differing substantially only in the intended treatment target.In both classes of treatment, it is the photoactivation of the pigmentthat is fundamentally responsible for the desired therapeutic outcome.

Accordingly, a preferred embodiment is shown in FIG. 5. The source 80produces a beam of light 82 consisting of a rapid series of high peakpower pulses of NIR light. For example, standard commercially availablemode-locked titanium-sapphire lasers are capable of outputtingmode-locked pulses with durations <200 fs and pulse energies of about1-20 nJ at pulse repetition frequencies in excess of 75 MHz. This sourceproduces a quasi-continuous beam of light having a relatively lowaverage power (up to several Watts) but high peak power (on the order of100 kW) that is continuously tunable over a NIR wavelength band fromapproximately 690-1080 mn. The pulse train from the source 80constitutes a beam of light 82 that is easily focussed using standardoptical means, such as reflective or refractive optics 84. The focusedbeam 86 can then be directed into a tumor 88 or other localizedtreatment target.

Simultaneous two-photon photoactivation of the melanin, melaninprecursors, or other endogenous pigments will be substantially limitedto the focal zone 90 of the focused light beam 86 due to the highinstantaneous irradiance level that is only present at the focus.Furthermore, regardless of whether melanin, melanin precursors, oranother endogenous pigment is present in surrounding healthy tissue 92or skin 94, insignificant collateral photoactivation, photodamage orconversion into a phototoxic product will occur outside the focal zone90. This is a consequence of the non-linear relationship betweeninstantaneous optical power and simultaneous two-photon excitation,which limits significant excitation to the focal zone 90. Even ifmelanin, melanin precursors, or another endogenous pigment is presentoutside of the focal zone 90, excitation intensities are below thatnecessary to produce significant photoactivation.

The apparatus of the present invention can also include, for example, afocusing apparatus for focusing the light throughout a range of focallengths extending from a surface of the tissue to a depth substantiallybeyond the surface. The source of light and focusing apparatus cooperateto promote simultaneous two-photon excitation of the pigment atcontrollable locations throughout the volume of tissue.

By scanning the location of the focus of the beam 86 throughout thevolume of the tumor 88, complete photoactivation of the melanin, melaninprecursors, or other endogenous pigments into a phototoxic productthroughout the tumor 88 can be effected. This scanning action can beproduced by changing the position of the focus 86 relative to the tumor88, or by moving the tumor 88 relative to a stationary focus 86location. The quality of the focal region 90 of the focused light beam86 may be improved by pre-expanding the light beam 82, using a beamexpander or other device, prior to focusing using standard opticalmeans.

This scanning can be done, for example, by positioning a focus of a beamof light over a range of positions so that a focal plane of the lightbeam occurs at a site located between a surface of the tissue and apoint substantially beyond the tissue surface. As a result, treating theparticular volume of tissue may extend to penetrate deep within thetissue. This scanning can further include varying, while the beam oflight is extant, the radial position of the focal plane within thetissue, thereby to photoactivate the endogenous pigment at amultiplicity of positions between the tissue surface and a positionlocated substantially beyond the tissue surface.

The simultaneous two-photon photoactivation embodiment of the presentinvention has several variations for the treatment of topical tissues,as shown in FIG. 6 and in FIG. 7. For example, the non-damaging natureof focused NIR light, shown in FIG. 6, or of non-focused NIR light,shown in FIG. 7, allows photoactivation of melanin or other endogenouspigments at topical locations without risk to underlying or surroundingtissues.

Focused simultaneous two-photon photoactivation of melanin or otherendogenous pigments for topical therapy, as shown in FIG. 6, is effectedwhen a beam of light 82 from a source 80 is focused 86 onto a tumor 88or other localized treatment target using standard optical means, suchas reflective or refractive optics 84. In this manner, photoactivationof the melanin, melanin precursors, or other endogenous pigments into aphototoxic product occurs only at the focal zone 90. The surroundinghealthy tissue 92 and skin 94 are unaffected in this process, even ifthey also contain melanin, melanin precursors, or another endogenouspigment, since photoactivation is substantially limited to the focalzone 90. As described previously, a scanning action can be used toeffect photoactivation of the melanin, melanin precursor, or otherendogenous pigment into a phototoxic product throughout the volume ofthe tumor 88.

Non-focused simultaneous two-photon photoactivation of melanin, melaninprecursors, or other endogenous pigments for topical therapy, as shownin FIG. 7, is effected when a non-focused or expanded beam of light 96from a source 80 is directed onto a topical tumor 88 or other localizedtreatment target. This beam of light 96 may have a cross sectional areasmaller than, equal to, or larger than that of the tumor 88. Sincemelanin, melanin precursors, or other endogenous pigments are present insubstantially higher levels in the tumor 88, the therapeutic action willbe substantially limited to the volume of the tumor 88. Since the beamof light 96 is non-damaging to tissues that do not contain a significantconcentration of pigment, damage to surrounding healthy tissue 92 andskin 94 is avoided. This embodiment may be particularly useful when theexact location, size and shape of the tumor 88 are not known, or when itis otherwise undesirable to carefully control the location ofapplication of the beam of light 96, since careful control of thelocation of the beam of light 96 is not critical for successfuladministration of this therapeutic regime. When non-focused light isused, employment of extremely high peak power excitation sources, suchas Q-switched lasers or regeneratively amplified mode-locked lasers, maybe beneficial due to their exceptionally high peak radiant power (whichis in the GW range) that will thereby afford a high instantaneousirradiance over a large area.

A final related variation of this preferred embodiment for simultaneoustwo-photon photoactivation is shown in FIG. 8, where a non-focused orexpanded beam of light 96 from a source 80 is directed onto a tumor 88or other localized treatment target located below the skin's surface.This beam of light 96 may have a cross sectional area smaller than,equal to, or larger than that of the tumor 88. Since melanin, melaninprecursors, or other endogenous pigments are present in substantiallyhigher levels in a tumor 88, the therapeutic action will besubstantially limited to the volume of the tumor 88. Since the beam oflight 96 is non-damaging to tissues that do not contain a significantconcentration of pigment, damage to surrounding healthy tissue 92 andskin 94 is avoided. This embodiment may also be particularly useful whenthe exact location, size and shape of the tumor 88 are not known, orwhen it is otherwise undesirable to carefully control the location ofapplication of the beam of light 96, since careful control of thelocation of the beam of light 96 is not critical for successfuladministration of this therapeutic regime. As in the previousnon-focused embodiment, employment of extremely high peak powerexcitation sources may be beneficial due to their exceptionally highpeak radiant power and potential high instantaneous irradiance over alarge area.

Preferably, the simultaneous two-photon excitation will be produced byan ultrashort pulsed NIR laser light having a wavelength of fromapproximately 450 nm to 1400 nm with a pulse width of from approximately25 fs to 10 ns and a greater than approximately 1 kHz pulse repetitionfrequency. Such laser light can be produced by a mode-lockedtitanium:sapphire laser or related laser sources.

The extent and duration of excitation affected with such sources will becontrolled by varying the location, irradiance and duration ofapplication of the light.

The effectiveness of the therapeutic outcome may be markedly increasedby simultaneous photoactivation and localized heating (hyperthermia) ofthe treatment site. Such heating occurs as a secondary effect ofillumination with laser light, and may also be controlled by varying thelocation, irradiance and duration of application of the light, so as toyield heating in the treatment zone of 2-10° C. above normaltemperatures. For example, application of light at intensities of150-3000 mW/cm² may be used to produce such desirable hyperthermia.Alternately, secondary thermal sources, such as infrared lamps or warmfluid baths, may be used to effect such desirable hyperthermia at thetreatment site.

While the foregoing disclosure has primarily focused on exampletherapeutic applications using two-photon excitation of agents withultrashort pulsed NIR light produced by mode-locked titanium:sapphirelasers, the present invention is not limited to such excitation nor tosuch narrowly defined optical sources. In fact, aspects of the presentinvention are applicable when optical excitation is effected usinglinear or other non-linear methods. For example, various other opticalsources are applicable, alone or in combination, such as continuous waveand pulsed lamps, diode light sources, semiconductor lasers; other typesof gas, dye, and solid-state continuous, pulsed, or mode-locked lasers,including: argon ion lasers; krypton ion lasers; helium-neon lasers;helium-cadmium lasers; ruby lasers; Nd:YAG, Nd:YLF, Nd:YAP, Nd:YVO4,Nd:Glass, and Nd:CrGsGG lasers; Cr:LiSF lasers; Er:YAG lasers; F-centerlasers; Ho:YAG and Ho:YLF lasers; copper vapor lasers; nitrogen lasers;optical parametric oscillators, amplifiers and generators;regeneratively amplified lasers; chirped-pulse amplified lasers; andsunlight.

In particular, of the aforementioned light sources, the regenerativelyamplified mode-locked lasers, such as the Coherent RegA-9000regeneratively amplified titanium:sapphire laser (which has a maximumspecified pulse energy of 4 μJ) and the Coherent Libra regenerativelyamplified titanium:sapphire laser (which has a maximum specified pulseenergy of approximately 1 mJ) are particularly useful for non-linearexcitation of endogenous or exogenous pigments, since such lasers affordan ultrashort pulsed output having a pulse energy of greater than about1 μJ and less than about 1 mJ. As such, these lasers are well suited toprovide the appropriate pulsed excitation necessary to efficiently andselectively excite pigments so that the pigment becomes photochemicallyactivated in a particular volume of tissue. For example, pulse energiessubstantially less than 1 μJ are generally insufficiently energetic toactivate pigments while energies in excess of 1 mJ are excessivelyenergetic, and can yield non-specific activation of tissue (i.e.,ablation of both pigmented and non-pigmented tissue). In contrast, theinventors of the present invention have found that energies in the rangeof greater than about 1 μJ and less than about 1 mJ, such as thoseproduced by regeneratively amplified titanium:sapphire lasers, areeffective for selective excitation of endogenous or exogenous pigments.

In an alternative embodiment, an exogenous photodynamic agent can beadded to the patient to be activated in conjunction with the endogenouspigments. “Exogenous” agents are photoactive materials not pre-existentin a patient or other target which are for example administered for thepurpose of increasing efficiency of conversion of optical energy into atherapeutic process. Examples of such exogenous agents include RoseBengal, psoralen derivatives, indocyanine, Lutex, Sn(ET₂) and variousporphyrin derivatives, including porfimer sodium and benzoporphyrinderivative. Preferably, the targeted tissue is pretreated with theexogenous agent so that it retains a therapeutic concentration of theagent when the tissue is treated with light so as to promotesimultaneous two-photon activation of the agent. Alternatively, theagent can be added at other times during the process. Uponadministration and accumulation in targeted tissue, such agents can beused to efficiently interact with NIR light so as to kill tissue by TypeI or Type II PDT mechanisms. Such killing can be used to augment orsupplement killing of pigmented tissues using endogenous photoactiveagents as described supra.

Another alternate embodiment of the present invention is directed to thethermal destruction of melanomas and other pigmented lesions.

Melanomas are usually dramatically darker than surrounding healthytissue. The dark color associated with melanomas is caused by increasedproduction of melanin by tumor cells. Melanin is a strong absorber ofultraviolet (UV) and visible light, and normally protects cells from thedeleterious effects of solar UV radiation. For example, FIG. 2 showsthat melanin is highly absorptive at wavelengths shorter thanapproximately 1000 nm. In contrast, hemoglobin has minimal absorbanceabove 450 nm. The high concentration of melanin in most melanoma cellsmakes them capable of strongly and selectively absorbing light atwavelengths longer than 450 nm and shorter than 1000 nm. Thus,illumination of melanoma cells with light at such wavelengths willproduce much more heat in those cells as compared to cells in lesspigmented tissue.

Currently, laser illumination is used in cosmetic applications to removeunwanted hair. Laser hair removal is accomplished because there is morepigment in the hair follicles than in surrounding tissue. Therefore,when a laser illuminates the pigmented hair follicle, it absorbs muchmore of the light, causing localized heating. The localized hyperthermiathereby created in the bulb of the hair follicle kills the hair folliclewhile sparing surrounding tissue (which is not heated to a significantextent by the laser illumination).

The inventors of the present application have discovered a process tokill pigmented tumor cells by thermally overloading them whereas therelatively unpigmented cells in healthy tissues surrounding the tumorare spared. FIGS. 9 and 10 illustrate such an alternate embodiment forthe present invention wherein a focused light beam 86 (FIG. 9) and anon-focused light beam 96 (FIG. 10), respectively, are used to killpigmented tumor cells 98. Such pigmented tumor cells 98 may be locatedat the surface of tissue 92 to be treated, or may be locatedsignificantly below the surface. Illumination of pigmented tumor cells98 may be effected using a continuous wave or pulsed laser sourceoperating in either of two wavelength bands between approximately 450and 800 nm and between approximately 800 and 1400 nm.

For wavelengths between 450 and 800 nm, direct linear excitation ofmelanin is used to selectively promote thermal overload of pigmentedtumor cells 98. Light in this band is preferred when pigmented tumorcells 98 are located at the surface of tissue or at depths ofapproximately 2 mm or less below the surface since such light is notcapable of penetrating tissue to significantly greater depths. For suchexcitation, it is preferred that illumination be effected viaapplication of one or more short pulses of light having a pulse durationof 10 ns (nanoseconds) or less, and more preferably of 10 ps(picoseconds) or less. Use of such short duration pulses reduces thermalloss to surrounding tissues, thereby improving efficiency in selectivethermal overload of the pigmented tumor cells 98. It is furtherpreferred that the wavelength of this light be between approximately 600and 800 nm to afford improved specificity for excitation of melaninrelative to hemoglobin. Moreover, it is further preferred that suchlight be produced by a light source such as a mode-lockedtitanium:sapphire laser, which is readily able to deliver such lightpulses at such wavelengths. A focused light beam 86 is preferable wherethe location and extent of the lesion is precisely known, since improvedcontrol over the extent of the treatment zone is thereby possible. Byscanning this focused light beam 86 throughout the volume of the tumor,it is possible to treat the entirety of the pigmented tumor cells 98.However, where the location and extent of the lesion is not preciselyknown, or where the lesion is exceptionally large, use of a non-focusedlight beam 96 is preferred to assure that treatment is effected in allof the pigmented tumor cells 98.

For wavelengths between 800 and 1400 nm, excitation of melanin vialinear mechanisms and non-linear two-photon mechanisms is used toselectively promote thermal overload of pigmented tumor cells 98. Lightin this band is preferred when pigmented tumor cells 98 are locatedbelow the surface of tissue at depths of approximately 2 mm or greatersince such light is capable of penetrating tissue to such depths. Forsuch excitation, it is preferred that illumination be effected viaapplication of one or more short pulses of light having a pulse durationof 10 ps or less, and more preferably of 1 ps or less. Use of such shortduration pulses increases the efficiency of non-linear excitationmechanisms while simultaneously reducing thermal loss to surroundingtissues, thereby improving efficiency in selective thermal overload ofthe pigmented tumor cells 98. A focused light beam 86 is preferablewhere the location and extent of the lesion is precisely known, sinceimproved control over the extent of the treatment zone is therebypossible. Use of such a focused light beam 86 improves efficiency ofnon-linear excitation mechanisms, allowing relatively low energy lightsources 80, such as mode-locked titanium:sapphire lasers, to besuccessfully used. By scanning this focused light beam 86 throughout thevolume of the tumor it is possible to treat the entirety of thepigmented tumor cells 98. However, where the location and extent of thelesion is not precisely known, or where the lesion is exceptionallylarge, use of a non-focused light beam 96 is preferred to assure thattreatment is effected in all of the pigmented tumor cells 98. Under suchillumination conditions, amplified or other higher energy light sources80, such as the regeneratively amplified mode-locked titanium:sapphirelaser, are preferred so as to increase illumination intensities tolevels sufficient to achieve efficient non-linear excitation.

In a further alternate embodiment for promoting thermal overload ofpigmented cells or volume of pigmented tissue, it is preferred that asingle beam of light be applied to such cells or volume of tissue,wherein such light is applied to such cells or volume of tissuecontinuously for a duration of at least 10 microseconds in order toachieve sufficient thermal overload. It is further preferred that suchcontinuous light application does not continue for more that about 1millisecond in order to avoid excessive thermal loss to surroundingtissues. Such temporal delivery may be achieved, for example, by use ofa step-scan or continuously scanned beam, so as to sequentiallyilluminate a broad area of tissue, or by pulsed delivery of light to aconfined or broad area of tissue.

It will be clear that the methods and apparatus described for thisalternate embodiment will be equally applicable to the treatment ofother pigmented blemishes, such as for example moles, port wine stains,freckles, scars, and tattoos, and for the reduction or elimination ofpigments in hair.

While the present invention has been illustrated and described asembodied in general methods and apparatus for killing pigmented tumorsby activation of endogenous pigments using optical radiation, it is notintended to be limited to the details shown, since it will be understoodthat various omissions, modifications, substitutions and changes in theforms and details of the method illustrated and in its operation can bemade by those skilled in the art without departing in any way from thespirit of the present invention.

This description has been offered for illustrative purposes only and isnot intended to limit the invention of this application, which isdefined in the claims below.

What is claimed as new and desired to be protected by Letters Patent isset forth in the appended claims.

1. A method for the treatment of a particular volume of tissue, saidvolume of tissue containing an endogenous pigment, the method comprisingthe steps of: non-invasively treating the particular volume of tissue byscanning with a beam of light to promote thermal overload of pigmentedcells in the particular volume of tissue, wherein said thermal overloadkills said pigmented cells, wherein said light is produced by a singlelight source, and wherein said light is applied to said volume of tissuecontinuously for a duration of at least 10 microseconds.
 2. The methodof claim 1 wherein the light to promote said thermal overload is a laserlight produced by a laser.
 3. The method of claim 2 wherein the laserlight comprises a train of one or more pulses.
 4. The method of claim 2including operating the laser to produce light at a wavelength betweenapproximately 450 nm to 800 nm.
 5. The method of claim 4 wherein saidwavelength of light is between approximately 600 nm and 800 nm.
 6. Themethod of claim 4 wherein said particular volume of tissue is locatedsubstantially at the tissue surface.
 7. The method of claim 4 whereinsaid particular volume of tissue is located approximately 2 mm or lessbelow the tissue surface.
 8. The method of claim 2 including operatingthe laser to produce light at a wavelength between approximately 800 nmto 1400 nm.
 9. The method of claim 8 wherein said particular volume oftissue is located approximately 2 mm or greater below the tissuesurface.
 10. The method of claim 1 wherein the light to promote saidthermal overload is a focused beam of light.
 11. The method of claim 10wherein the focused beam of light is focused laser light.
 12. The methodof claim 1 wherein the light to promote said thermal overload is anon-focused beam of light.
 13. The method of claim 1 wherein saidendogenous pigment is selected from the group comprising melanin,melanin precursors, carotenes, porphyrins, and various tattoo dyes. 14.The method of claim 13 wherein said melanin precursors are selected fromthe group comprising 5-S-cysteinyldopa (5-SCD) and 5,6-dihidroxyindole(DHI), dopa, dopa semiquinone, leucodopachrome, dopachrome, eumalanins,pheomelanins, sepia melanins, and 5,6-dihydroxyindole-2-carboxylic acid.15. The method of claim 13 wherein said porphyrins include hemoglobin.